Skip to main content
SpringerLink
Log in

Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane

  • Food Biotechnology & Probiotics - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Agcam E, Akyildiz A, Akdemir EG (2014) Comparison of phenolic compounds of orange juice processed by pulsed electric fields (PEF) and conventional thermal pasteurisation. Food Chem 143:354–361. https://doi.org/10.1016/j.foodchem.2013.07.115

    Article  CAS  PubMed  Google Scholar 

  2. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408

    Article  CAS  PubMed  Google Scholar 

  3. Chen B, Fan DQ, Zhu KX, Shan ZG, Chen FY, Hou L, Cai L, Wang KJ (2015) Mechanism study on a new antimicrobial peptide Sphistin derived from the N-terminus of crab histone H2A identified in haemolymphs of Scylla paramamosain. Fish Shellfish Immunol 47:833–846. https://doi.org/10.1016/j.fsi.2015.10.010

    Article  CAS  PubMed  Google Scholar 

  4. Chopra L, Singh G, Kumar JK, Sahoo DK (2015) Sonorensin: a new bacteriocin with potential of an anti-biofilm agent and a food biopreservative. Sci Rep 5:13412. https://doi.org/10.1038/srep13412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71:1–20

    Article  CAS  PubMed  Google Scholar 

  6. Cotter PD, Ross RP, Hill C (2013) Bacteriocins—a viable alternative to antibiotics? Nat Rev Microbiol 11:95–105. https://doi.org/10.1038/nrmicro2937

    Article  CAS  PubMed  Google Scholar 

  7. Eijsink VG, Axelsson L, Diep DB, Havarstein LS, Holo H, Nes IF (2002) Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Van Leeuwenhoek 81:639–654

    Article  CAS  PubMed  Google Scholar 

  8. Gharsallaoui A, Oulahal N, Joly C, Degraeve P (2016) Nisin as a food preservative: part 1: physicochemical properties, antimicrobial activity, and main uses. Crit Rev Food Sci Nutr 56:1262–1274. https://doi.org/10.1080/10408398.2013.763765

    Article  CAS  PubMed  Google Scholar 

  9. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp 41:95–98

    CAS  Google Scholar 

  10. Hoffmann M, Luo Y, Monday SR, Gonzalez-Escalona N, Ottesen AR, Muruvanda T, Wang C, Kastanis G, Keys C, Janies D, Senturk IF, Catalyurek UV, Wang H, Hammack TS, Wolfgang WJ, Schoonmaker-Bopp D, Chu A, Myers R, Haendiges J, Evans PS, Meng J, Strain EA, Allard MW, Brown EW (2016) Tracing Origins of the Salmonella bareilly strain causing a food-borne outbreak in the United States. J Infect Dis 213:502–508. https://doi.org/10.1093/infdis/jiv297

    Article  PubMed  Google Scholar 

  11. Hultman J, Rahkila R, Ali J, Rousu J, Bjorkroth KJ (2015) Meat processing plant microbiome and contamination patterns of cold-tolerant bacteria causing food safety and spoilage risks in the manufacture of vacuum-packaged cooked sausages. Appl Environ Microbiol 81:7088–7097. https://doi.org/10.1128/AEM.02228-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hurdle JG, O’Neill AJ, Chopra I, Lee RE (2010) Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Microbiol 9:62

    Article  Google Scholar 

  13. Joerger MC, Klaenhammer TR (1990) Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin. J Bacteriol 172:6339–6347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kadariya J, Smith TC, Thapaliya D (2014) Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int 2014:827965. https://doi.org/10.1155/2014/827965

    Article  PubMed  PubMed Central  Google Scholar 

  15. Lavieri NA, Sebranek JG, Cordray JC, Dickson JS, Horsch AM, Jung S, Manu DK, Mendonça AF, Stecher BB (2015) Control of Listeria monocytogenes on alternatively cured ready-to-eat ham using natural antimicrobial ingredients in combination with post-lethality interventions. Food Process Technol 6:10

    Google Scholar 

  16. Lazdunski CJ (1995) Colicin import and pore formation: a system for studying protein transport across membranes? Mol Microbiol 16:1059–1066

    Article  CAS  PubMed  Google Scholar 

  17. Lee H, Kim HY (2011) Lantibiotics, class I bacteriocins from the genus Bacillus. J Microbiol Biotechnol 21:229–235

    CAS  PubMed  Google Scholar 

  18. Li J, Ahn J, Liu D, Chen S, Ye X, Ding T (2016) Evaluation of ultrasound-induced damage to Escherichia coli and Staphylococcus aureus by flow cytometry and transmission electron microscopy. Appl Environ Microbiol 82:1828–1837. https://doi.org/10.1128/AEM.03080-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Liu G, Song Z, Yang X, Gao Y, Wang C, Sun B (2016) Antibacterial mechanism of bifidocin A, a novel broad-spectrum bacteriocin produced by Bifidobacterium animalis BB04. Food Control 62:309–316

    Article  CAS  Google Scholar 

  20. Liu YG, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43:649–650, 652, 654

  21. Lou M, Zhu B, Muhammad I, Li B, Xie G, Wang Y, Li H, Sun G (2011) Antibacterial activity and mechanism of action of chitosan solutions against apricot fruit rot pathogen Burkholderia seminalis. Carbohyd Res 346:1294–1301. https://doi.org/10.1016/j.carres.2011.04.042

    Article  CAS  Google Scholar 

  22. Lou MM, Zhu B, Muhammad I, Li B, Xie GL, Wang YL, Li HY, Sun GC (2011) Antibacterial activity and mechanism of action of chitosan solutions against apricot fruit rot pathogen Burkholderia seminalis. Carbohydr Res 346:1294–1301. https://doi.org/10.1016/j.carres.2011.04.042

    Article  CAS  PubMed  Google Scholar 

  23. Mercer RG, Walker BD, Yang X, McMullen LM, Ganzle MG (2017) The locus of heat resistance (LHR) mediates heat resistance in Salmonella enterica, Escherichia coli and Enterobacter cloacae. Food Microbiol 64:96–103. https://doi.org/10.1016/j.fm.2016.12.018

    Article  CAS  PubMed  Google Scholar 

  24. Moll GN, Konings WN, Driessen AJ (1999) Bacteriocins: mechanism of membrane insertion and pore formation. Antonie Van Leeuwenhoek 76:185–198

    Article  CAS  PubMed  Google Scholar 

  25. NicAogain K, O’Byrne CP (2016) The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain. Front Microbiol 7:1865. https://doi.org/10.3389/fmicb.2016.01865

    Article  PubMed  PubMed Central  Google Scholar 

  26. Nigutova K, Serencova L, Piknova M, Javorsky P, Pristas P (2008) Heterologous expression of functionally active enterolysin A, class III bacteriocin from Enterococcus faecalis, in Escherichia coli. Protein Expr Purif 60:20–24. https://doi.org/10.1016/j.pep.2008.03.006

    Article  CAS  PubMed  Google Scholar 

  27. Nilsen T, Nes IF, Holo H (2003) Enterolysin A, a cell wall-degrading bacteriocin from Enterococcus faecalis LMG 2333. Appl Environ Microbiol 69:2975–2984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Oppegard C, Rogne P, Emanuelsen L, Kristiansen PE, Fimland G, Nissen-Meyer J (2007) The two-peptide class II bacteriocins: structure, production, and mode of action. J Mol Microbiol Biotechnol 13:210–219. https://doi.org/10.1159/000104750

    Article  CAS  PubMed  Google Scholar 

  29. Ozogul F, Hamed I (2017) The importance of lactic acid bacteria for the prevention of bacterial growth and their biogenic amines formation: a review. Crit Rev Food Sci Nutr. https://doi.org/10.1080/10408398.2016.1277972

    PubMed  Google Scholar 

  30. Padmavathy N, Vijayaraghavan R (2008) Enhanced bioactivity of ZnO nanoparticles-an antimicrobial study. Sci Technol Adv Mater 9:35004. https://doi.org/10.1088/1468-6996/9/3/035004

    Article  Google Scholar 

  31. Pillet F, Formosa-Dague C, Baaziz H, Dague E, Rols MP (2016) Cell wall as a target for bacteria inactivation by pulsed electric fields. Sci Rep 6:19778. https://doi.org/10.1038/srep19778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Riley MA (1993) Molecular mechanisms of colicin evolution. Mol Biol Evol 10:1380–1395

    CAS  PubMed  Google Scholar 

  33. Schenk M, Raffellini S, Guerrero S, Blanco GA, Alzamora SM (2011) Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae by UV-C light: study of cell injury by flow cytometry. Lebensmittel-Wissenschaft und-Technologie 44:191–198

    Article  CAS  Google Scholar 

  34. Sun Z, Kong J, Hu S, Kong W, Lu W, Liu W (2013) Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen. Appl Microbiol Biotechnol 97:1941–1952. https://doi.org/10.1007/s00253-012-4044-x

    Article  CAS  PubMed  Google Scholar 

  35. Sung SY, Sin LT, Tee TT, Bee ST, Rahmat AR, Rahman WAWA, Tan AC, Vikhraman M (2013) Antimicrobial agents for food packaging applications. Trends Food Sci Tech 33:110–123

    Article  CAS  Google Scholar 

  36. Hp T, Oppedijk SF, Breukink E, Martin NI (2016) New insights into Nisin’s antibacterial mechanism revealed by binding studies with synthetic lipid II analogues. Biochemistry-Us 55:232–237. https://doi.org/10.1021/acs.biochem.5b01173

    Article  Google Scholar 

  37. Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinform Chapter 2:2–3. https://doi.org/10.1002/0471250953.bi0203s00

    Google Scholar 

  38. Thompson JK, Collins MA, Mercer WD (1996) Characterization of a proteinaceous antimicrobial produced by Lactobacillus helveticus CNRZ450. J Appl Bacteriol 80:338–348

    Article  CAS  PubMed  Google Scholar 

  39. Tsiraki MI, Savvaidis IN (2016) The effects of citrus extract (Citrox(c)) on the naturally occurring microflora and inoculated pathogens, Bacillus cereus and Salmonella enterica, in a model food system and the traditional Greek yogurt-based salad Tzatziki. Food Microbiol 53:150–155. https://doi.org/10.1016/j.fm.2015.09.015

    Article  PubMed  Google Scholar 

  40. Vaara M (1992) Agents that increase the permeability of the outer membrane. Microbiol Rev 56:395

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Woraprayote W, Malila Y, Sorapukdee S, Swetwiwathana A, Benjakul S, Visessanguan W (2016) Bacteriocins from lactic acid bacteria and their applications in meat and meat products. Meat Sci 120:118–132. https://doi.org/10.1016/j.meatsci.2016.04.004

    Article  CAS  PubMed  Google Scholar 

  42. Zhang M, Wang D, Geng Z, Li P, Sun Z, Xu W (2018) Effect of heat shock protein 90 against ROS-induced phospholipid oxidation. Food Chem 240:642–647. https://doi.org/10.1016/j.foodchem.2017.08.005

    Article  CAS  PubMed  Google Scholar 

  43. Zhang T, Pan Y, Li B, Ou J, Zhang J, Chen Y, Peng X, Chen L (2013) Molecular cloning and antimicrobial activity of enterolysin A and helveticin J of bacteriolysins from metagenome of Chinese traditional fermented foods. Food Control 31:499–507

    Article  CAS  Google Scholar 

  44. Zhao W, Yang R, Zhang HQ, Zhang W, Hua X, Tang Y (2011) Quantitative and real time detection of pulsed electric field induced damage on Escherichia coli cells and sublethally injured microbial cells using flow cytometry in combination with fluorescent techniques. Food Control 22:566–573

    Article  Google Scholar 

  45. Zou Y, Bian H, Li P, Sun Z, Sun C, Zhang M, Geng Z, Xu W, Wang D (2017) Optimization and physicochemical properties of nutritional protein isolate from pork liver with ultrasound-assisted alkaline extraction. Anim Sci J. https://doi.org/10.1111/asj.12930

    Google Scholar 

Download references

Acknowledgements

This study was supported by National Natural Science Foundation of China (31601530), the Natural Science Foundation of Jiangsu Province (BK20151367), Innovation of Agricultural Science and Technology of Jiangsu Province (CX(15)1008) and China agriculture research system (CARS-41).

Author information

Authors and Affiliations

  1. Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, People’s Republic of China

    Zhilan Sun, Xiaomeng Wang, Xinxiao Zhang, Haihong Wu, Ye Zou, Pengpeng Li, Chong Sun, Weimin Xu, Fang Liu & Daoying Wang

  2. Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, Nanjing, 210095, People’s Republic of China

    Weimin Xu

Authors
  1. Zhilan Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaomeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xinxiao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Haihong Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ye Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pengpeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chong Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Weimin Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Fang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daoying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Fang Liu or Daoying Wang.

Ethics declarations

Conflict of interest

The authors declare no competing financial interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, Z., Wang, X., Zhang, X. et al. Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane. J Ind Microbiol Biotechnol 45, 213–227 (2018). https://doi.org/10.1007/s10295-018-2008-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-018-2008-6

Keywords

Search

Navigation